Nitrogen fixation, the reduction of atmospheric dinitrogen to ammonia catalyzed by the enzyme nitrogenase, is the sole biological process for replenishing the nitrogen that is used in the biosynthesis of cellular materials. This enzyme system consists of two metalloproteins, the Fe-protein and MoFe-protein, that mediate the coupling of ATP hydrolysis to substrate reduction. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. We will utilize crystallographic, biochemical and spectroscopic approaches to investigate the enzymatic and metallocluster assembly mechanisms of nitrogenase. Special emphasis will be placed on establishing the atomic identity and mechanistic significance of the light-atom ligand we recently observed in the center of the FeMo-cofactor, and in assessing the structural framework for the nucleotide-mediated gating of electron transfer processes in nitrogenase. Towards these objectives, we will address: 1. Very high resolution (<1.2A) structures of the nitrogenase proteins, and particularly the associated metalloclusters, in defined oxidation states. This will include not only establishing the metric parameters of the clusters, but also the use of diffraction-based methods to assign the oxidation states to individual metal sites within each cluster, and to determine the orientation of the relevant EPR g-tensors with respect to the metallocluster structures. Intermediates in the incorporation of the FeMo-cofactor into the MoFe-protein will also be studied to address aspects of the biosynthetic mechanism of the nitrogenase metalloclusters. 2. The interactions of Fe-protein with MgATP and the site(s) of binding to the physiological electron donors for the reduction of substrates, ferredoxin and flavodoxin. 3. The mode of substrate and inhibitor binding to nitrogenase under turnover conditions, initially through spectroscopic methods and ultimately by crystallography. From these studies, we seek to establish molecular details of mechanistically relevant states of the nitrogenase proteins that are essential to defining a chemically explicit mechanism for substrate reduction. Broader implications will address nucleotide dependent transduction processes based on the similarities between Fe-protein and other nucleotide switch proteins involved in signal and energy transduction processes.